OTULIN protects the intestinal epithelium from apoptosis during inflammation and infection

The intestinal epithelium is a single cell layer that is constantly renewed and acts as a physical barrier that separates intestinal microbiota from underlying tissues. In inflammatory bowel disease (IBD) in humans, as well as in experimental mouse models of IBD, this barrier is impaired, causing microbial infiltration and inflammation. Deficiency in OTU deubiquitinase with linear linkage specificity (OTULIN) causes OTULIN-related autoinflammatory syndrome (ORAS), a severe inflammatory pathology affecting multiple organs including the intestine. We show that mice with intestinal epithelial cell (IEC)-specific OTULIN deficiency exhibit increased susceptibility to experimental colitis and are highly sensitive to TNF toxicity, due to excessive apoptosis of OTULIN deficient IECs. OTULIN deficiency also increases intestinal pathology in mice genetically engineered to secrete excess TNF, confirming that chronic exposure to TNF promotes epithelial cell death and inflammation in OTULIN deficient mice. Mechanistically we demonstrate that upon TNF stimulation, OTULIN deficiency impairs TNF receptor complex I formation and LUBAC recruitment, and promotes the formation of the cytosolic complex II inducing epithelial cell death. Finally, we show that OTULIN deficiency in IECs increases susceptibility to Salmonella infection, further confirming the importance of OTULIN for intestinal barrier integrity. Together, these results identify OTULIN as a major anti-apoptotic protein in the intestinal epithelium and provide mechanistic insights into how OTULIN deficiency drives gastrointestinal inflammation in ORAS patients.


INTRODUCTION
The intestinal epithelium represents the largest surface area of the body in contact with the external environment. The epithelium is renewed every 4-5 days through a balanced process of stem cell proliferation and transit-amplifying cell proliferation in the intestinal crypts and, conversely, detachment and cell death of terminally differentiated enterocytes at the villus apex [1]. The single-cell layer of intestinal epithelial cells (IECs) and overlying mucus layer forms a physical barrier that protects the body from invasion by gut microbes. Excessive death of IECs impairs the integrity of this barrier and compromises intestinal homeostasis, as is shown in inflammatory bowel disease (IBD) and in several mouse models of intestinal pathology [2].
Tight regulation of cell death and NF-κB responses in the intestinal epithelium is important for intestinal homeostasis and pathology, as shown in many genetic mouse models [16][17][18][19][20][21][22][23][24] but also in patients with IBD [19,[25][26][27]. Since OTULIN is known to regulate NF-κB signaling and cell death responses [7,11], and since clinical manifestations in ORAS patients include intestinal abnormalities [9,10,12,28], we here investigated the role of OTULIN in the intestinal epithelium. For this, we generated mice that specifically lack OTULIN in IECs. These mice do not show a spontaneous phenotype but are more susceptible to experimental colitis induced by dextran sodium sulfate (DSS), and to intestinal infection with nontyphoidal Salmonella. These mice are also hypersensitive to a normally sublethal dose of TNF, which causes massive apoptosis of IECs inducing lethality. Consistent with this, OTULIN deficiency in IECs promotes the development of intestinal pathology in a chronic TNF-driven model of ileitis.

OTULIN IEC-KO mice are healthy and do not develop intestinal pathology
To investigate the role of OTULIN in the maintenance of the intestinal barrier, we generated mice that specifically lack OTULIN in the intestinal epithelium. To do this, mice with a floxed exon 3 of Otulin [14] were crossed with transgenic mice expressing Cre recombinase under control of the Villin promotor (Fig. 1A). The Villin-Cre transgenic line targets all epithelial cell lineages of the small intestine, cecum and colon, and expression mediates efficient Cre-mediated recombination in IECs starting before birth [29]. IEC-specific OTULIN knockout (Otulin FL/FL Villin-Cre, hereafter referred to as OTULIN IEC-KO ) mice were born with normal Mendelian segregation (Suppl. Fig. 1A). OTULIN deletion was confirmed on protein level both in IEC lysates from the small intestine of OTULIN IEC-KO mice as well as in small intestinal organoids derived from these mice (Fig. 1B, C). Western blot analysis also revealed that the expression levels of the LUBAC proteins HOIP, HOIL and SHARPIN are stable in IEC cells lacking OTULIN (Fig. 1B, C), which is similar to what was reported for bone marrow derived macrophages and THP-1 monocytes lacking OTULIN but different from what has been described for OTULINdeficient B cells, T cells, fibroblasts, hepatocytes and keratinocytes [9,10,[13][14][15]. Analysis of linear ubiquitination by specific UBAN pulldowns also confirmed the accumulation of linear polyubiquitin in IEC lysates of OTULIN-deficient mice (Fig. 1B), as previously shown in other OTULIN-deficient cell types [9,11,[13][14][15]30].
OTULIN IEC-KO mice look healthy and have normal body weight (Suppl. Fig. 1B, C), and phenotypic analysis of OTULIN IEC-KO mice up to the age of 12 months revealed no pathological signs in the intestine. Histologic analysis of tissue sections by hematoxylineosin staining showed normal morphology of both the small intestine and the colon of OTULIN IEC-KO mice (Fig. 1D). Also, no difference in the number of goblet cells and Paneth cells could be observed between wild-type (WT) and OTULIN IEC-KO mice, as revealed by Alcian-blue-Periodic acid Schiff (AB-PAS) staining (Fig.  1D). In agreement, expression analysis of Paneth cell-specific antimicrobial peptides (AMPs) Cryptidin-1, Lysozyme-P, Reg3γ and secreted PLA2 and of goblet cell-specific Muc2 by quantitative PCR on ileal epithelial cell lysates did not show any difference between WT and OTULIN IEC-KO mice (Suppl. Fig. 2A). Next, since OTULIN was shown to prevent cell death [10,11,[13][14][15]30], we performed an immunostaining for cleaved caspase-3 to detect apoptotic cells. However, no differences could be observed in the number of apoptotic enterocytes between WT and OTULIN IEC-KO mice, and only sporadically homeostatic apoptotic cells could be observed in the villus tip of the small intestines or at the top of the crypts in the colon of both lines (Fig. 1D). Also no differences were observed in expression of tight junction and desmosomal genes (ZO-1, Claudin-4, Occludin, Desmoglein) in intestinal lysates of WT and OTULIN IEC-KO mice (Suppl. Fig. 2B). In addition, no differences in epithelial cell proliferation could be observed between WT and OTULIN IEC-KO mice by Ki67 staining (Fig. 1D) genes (IκBα, Tnfaip3, Muc2, Cryptidin-1) in intestinal lysates of WT and OTULIN IEC-KO mice. However, no significant differences in expression could be observed between both genotypes (Suppl. Fig. 2A, C). Together, these data demonstrate that intestinal epithelial OTULIN is dispensable for intestinal homeostasis in steady state conditions.
OTULIN IEC-KO mice are sensitized to experimental colitis Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the intestine of which the etiology is multifactorial and incompletely understood. One factor that can contribute to IBD development is an unstable mucosal barrier, allowing microbial infiltration and hyperactive immune responses towards the commensal microbiota [31]. To address whether OTULIN expression determines the susceptibility to IBD, OTULIN IEC-KO mice and control WT littermates were evaluated in the established model of dextran sulfate sodium (DSS)-induced colitis. For this, mice were treated with 2% DSS in drinking water for 6 days followed by 8 days of regular drinking water, and were monitored daily for clinical pathology based on body weight loss, presence of fecal blood and stool consistency. Although OTULIN IEC-KO mice and WT littermate control mice showed a similar disease progression with maximal clinical colitis on day 8-9 ( Fig. 2A), OTULIN deficiency resulted in a slower recovery from colitis, as shown by the slower weight gain in the recovery phase of the experiment (Fig. 2B). However, no difference in colon length and colon histology could be detected (Fig. 2C, D), and no differences in the number of apoptotic cells could be observed by immunostaining for cleaved caspase-3 between WT and OTULI-N IEC-KO mice (Fig. 2E, F), at peak of disease (day 9) or at the end of the experiment (day 14). In conclusion, OTULIN IEC-KO mice recover slower from DSS-induced colitis, suggesting that OTULIN is needed to reestablish the intestinal barrier stability upon acute inflammation.
TNF induces IEC apoptosis and lethality in OTULIN IEC-KO mice TNF is a major proinflammatory cytokine that contributes to the pathogenesis of IBD in humans and experimental colitis in mice [26,32]. Since OTULIN has been shown to regulate TNF signaling [33], we evaluated the impact of OTULIN deficiency in IECs on TNFinduced pathology. For this, we injected OTULIN IEC-KO mice and control littermates with a normally sublethal dose of TNF. In contrast to control mice that only showed a modest drop in body temperature and all survived, OTULIN IEC-KO mice were hypersensitive to TNF and showed a steep drop in body temperature already 2 h after TNF injection, and all died three to 5 h after TNF injection (Fig. 3A). This was accompanied by severe inflammation, as shown by high concentrations of the proinflammatory cytokines and chemokines, IL-6 and Ccl2, in serum of OTULIN IEC-KO mice two and 3 h after TNF injection (Fig. 3B). Similarly, high expression of inflammatory mediators could be observed in IEC lysates of OTULIN IEC-KO mice (Suppl. Fig. 3A). Immune profiling of myeloid cell populations using flow cytometry and immunostaining for F4/ 80 positive cells did not reveal major differences in infiltration of neutrophils, monocytes, macrophages, dendritic cells and eosinophils in the mucosa of the small intestine between both genotypes after TNF challenge (Suppl. Fig. 3B-D). However, histological analysis of the ileum showed extensive epithelial destruction, massive villus atrophy and reduced goblet cell numbers in the OTULIN IEC-KO mice 4 h after TNF challenge, in contrast to control mice which maintain tissue integrity (Fig. 3C). We detected massive epithelial cell apoptosis both in the crypts and in the villi of OTULIN IEC-KO mice by cleaved caspase-3 and TUNEL staining, while only limited cell death could be observed in the small intestine of WT mice (Fig. 3C). Increased apoptosis was also observed in the colon of OTULIN IEC-KO mice after TNF injection, in contrast to the colon of WT mice where no apoptotic cells could be detected (Suppl. Fig. 4A, B). These observations suggest that TNF-driven breakdown of the intestinal barrier causes infiltration of commensal bacteria and systemic inflammation in OTULIN IEC-KO mice.
previously shown [8]. These alterations coincide with enhanced formation of a cytosolic complex comprised of FADD and caspase 8, which is more abundant in OTULIN deficient HCT116 cells treated with TNF alone or with TNF and zVAD (Fig. 4F). A Representative images of organoids from the small intestine isolated from WT and OTULIN IEC-KO mice which were left untreated or stimulated with 20 ng/mL mTNF for the indicated time points. Viability was assessed by caspase-3/7 green detection reagent. Scale bar, 100 μm. n = 3 biological replicates for each group and three technical replicates for each biological replicate. B Quantification of cell death as measured by caspase-3/7-positive signal in organoid cultures from WT and OTULIN IEC-KO mice either or not stimulated with 20 ng/ml mTNF (n = 3 biological replicates for each group and three technical replicates for each biological replicate). Data are represented as mean ± SEM, *p < 0.05. Statistics represent differences in cell death between the genotypes over time. C Western blot analysis for full length (FL) and cleaved (CL) caspase-3 in lysates of small intestinal organoids isolated from OTULIN IEC-KO (KO, n = 3 biological replicates and WT, n = 1) mice either stimulated or not stimulated with 20 ng/mL mTNF for 6 h. Actin was used as a loading control. D WT and OTULIN KO HCT116 cells were treated with hTNF (20 ng/mL) for 24 h after which cell death was analysed by flow cytometry using propidium iodide and annexin V staining. Within the same condition, each dot is the result of a different experiment and is the mean of technical duplicates. **p < 0.01; ***p < 0.001. E WT and OTULIN KO HCT116 cells were treated with 1.5 µg/ml FLAG-hTNF for the indicated timepoints. TNFR1 complex I was FLAG-immunoprecipitated and followed by USP21 treatment on the IPs. Protein levels were determined by immunoblotting. Actin was used as a loading control. Representative of two independent experiments. F WT and OTULIN KO HCT116 cells were pretreated for 30 min with 50 µM ZVAD, as indicated, followed by 20 ng/mL hTNF for 4 h. RIPK1 was immunoprecipitated and protein levels were determined by immunoblotting. Actin was used as a loading control. Representative of two independent experiments. only minor differences could be observed in TNF-induced NF-κB and MAPK signaling between control and OTULIN deficient HCT116 cells (Suppl. Figure 5).
Together, these data demonstrate that OTULIN is critical to prevent IEC apoptosis in conditions of acute TNF exposure.

OTULIN deficiency increases intestinal pathology in TNF emARE mice
To investigate the importance of OTULIN in conditions of chronic TNF exposure, we crossed OTULIN IEC-KO mice with TNF emARE mice. TNF emARE mice were generated by CRISPR/Cas9-mediated genome engineering, and lack the AU-rich elements (ARE) in the Tnf gene, leading to stabilization of Tnf mRNA and chronic elevated levels of TNF in the mice. Heterozygous TNF emARE/+ mice do not develop any spontaneous pathology, in contrast to homozygous TNF emARE/ARE mice (unpublished data, Thiran et al., EMBO Mol. Med., in press), which phenocopy the previously published TNF ΔARE mouse line [35] that spontaneously develop arthritis and ileitis. We confirmed the increased concentration of TNF in the serum of TNF emARE/+ mice compared to wild-type littermates, but TNF levels were not further increased upon OTULIN deletion (Fig. 5A). IEC-specific OTULIN deficiency did not affect body weight in TNF emARE/+ mice (Fig. 5B). However, histological analysis showed increased intestinal pathology in the jejunum of OTULIN IEC-KO TNF emARE/+ mice compared to TNF emARE/+ mice (Fig. 5C, D). In contrast, no difference in disease development was observed in the ileum (Fig. 5C, D). Although cleaved caspase-3-positive apoptotic cells could be detected in the villi of TNF emARE/+ mice, significantly more cell death could be observed both in the crypts and the villi of the jejunum of OTULIN IEC-KO TNF emARE/+ mice (Fig. 5C, E). A similar trend could be observed in the ileum of OTULIN IEC-KO TNF emARE/+ mice compared to TNF emARE/+ mice, although not significant (p-value: 0.06) (Fig. 5C, E). Finally, we also addressed intestinal pathology in the colon, and observed increased numbers of cleaved caspase-3-positive apoptotic cells in OTULIN IEC-KO TNF emARE/+ colon compared to the TNF emARE/+ condition (Fig. 5F, G).
Together, these data show that OTULIN also critically controls TNF-induced cell death in a transgenic model of chronic TNFdriven intestinal inflammation.
OTULIN IEC-KO mice are sensitized to Salmonella-induced colitis To complement our genetic and exogenous TNF supplementation models of OTULIN-dependent sensitivity, we evaluated colitis development in response to infection with the Salmonella enterica serovar Typhimurium (Salmonella) [36]. Salmonella infection induces TNF expression and hence provides an endogenous source of TNF within the intestinal tract [37]. Further, Salmonella infection leads to intestinal epithelial cell apoptosis, which acts as a nutrient source for bacteria [38]. OTULIN IEC-KO and littermate control mice exhibited similar systemic morbidity during acute Salmonella infection as both genotypes lost body weight to a similar degree (Fig. 6A), and OTULIN IEC-KO mice had similar cecal and spleen weights, and inflammatory gene expression in IECs as littermate controls (Suppl. Fig. 6A-C). However, OTULIN IEC-KO mice showed a reduced colon length (Fig. 6B), suggesting that OTULIN deficiency might impact disease at the colonic tissue level. To assess if OTULIN-deficiency influenced cell death during Salmonella infection, we stained proximal and distal colon sections for cleaved caspase-3 or TUNEL. There was an increase in both apoptotic and general cell death, particularly in the distal colons of  OTULIN IEC-KO mice (Fig. 6C, D). Increased cell death correlated with exaggerated Salmonella burden in the colon and in bacterial dissemination to the spleen (Fig. 6E) but not in the small intestine or cecum (Suppl. Fig. 6D). Salmonella uses two type-3 secretion systems encoded within the Salmonella pathogenicity island 1 (SPI-1) and SPI-2 that are largely responsible for inflammation and mammalian cell death in this model [38]. To test if OTULIN-dependent sensitivity requires these Salmonella virulence factors, we infected WT and OTULIN IEC-KO mice with a mutant ΔSPI-1ΔSPI-2 strain. In contrast to fully virulent Salmonella, ΔSPI-1ΔSPI-2 Salmonella does not induce body weight loss (Fig. 6F). In the absence of SPI-1/SPI-2, OTULIN IEC-KO mice had  Fig. 6E, F), did not show shortened colon length (Fig. 6G), nor increases in epithelial cell death (Fig. 6H, Suppl. Fig. 6G), or increased Salmonella burden compared to littermate controls (Fig. 6I, Suppl. Fig. 6H). Together, these findings demonstrate that OTULIN signaling within intestinal epithelial cells is critical for restricting cell death and pathogen burden in response to inflammation-inducing virulent Salmonella infection.

DISCUSSION
Lytic cell death is increasingly recognized as a major driver of inflammatory pathologies. Lytic forms of cell death, including necroptosis, pyroptosis, and secondary necrosis release DAMPs (danger-associated molecular patterns), that activate immune receptors and induce inflammatory responses. In contrast, apoptosis is commonly considered an anti-inflammatory cell death modality in most tissues, but excessive apoptosis in the intestinal epithelium can nonetheless promote inflammation due to loss of barrier integrity and the subsequent sensing of PAMPs (pathogen-associated molecular patterns) from commensal microbes that have breached the barrier [39]. Cells have developed multiple mechanisms to keep cell death in check to prevent the development of chronic inflammation, particularly at barrier sites. OTULIN has been identified as a critical regulator of cell death and inflammation in different organs such as liver and skin [11,[13][14][15]30]. Humans with a homozygous loss-of-function mutation in OTULIN develop OTULIN-related autoinflammatory syndrome (ORAS), a potentially fatal, TNF-driven autoinflammatory disease characterized by severe systemic inflammation, recurrent fevers, panniculitis, and arthritis, but also with diarrhea [9,10,12], suggesting a role for OTULIN in intestinal homeostasis. Although deletion of OTULIN in the intestinal epithelium of mice did not cause spontaneous intestinal pathology, we showed that these mice are hypersensitive to a TNF challenge, causing severe epithelial destruction and lethality. Indeed, OTULIN deficient IECs are shown to die from TNF-induced apoptosis. ORAS patients have high levels of circulating TNF with effects on multiple organs. To model the chronic systemic TNF condition in ORAS patients, we generated IEC-specific OTULIN deficient mice in the TNF emARE/+ background which spontaneously produce elevated levels of TNF. In response to chronic elevated TNF exposure, OTULIN IEC-KO TNF emARE/+ mice develop elevated intestinal pathology and display enhanced epithelial cell death in the intestine. Analysis of intestinal biopsies of ORAS patients could reveal whether intestinal pathology in these patients is associated with increased IEC cell death.
The function of OTULIN is likely to prevent accumulation of M1ubiquitin chains in the cytoplasm and prevent LUBAC autoubiquitination and degradation [8,11]. Hence, OTULIN-deficient cells often show reduced expression of HOIP and SHARPIN, with enhanced sensitization to TNF-induced cell death [10,11,15,30]. The intestinal epithelium of OTULIN IEC-KO mice, however, shows normal expression levels of LUBAC proteins, questioning the mechanism of how OTULIN deficiency promotes epithelial cell death. Using HCT116 cells we demonstrated that, although LUBAC levels are unaffected in basal conditions, its recruitment to the TNF receptor signaling complex I in response to TNF stimulation is significantly impaired in OTULIN deficient conditions, promoting the formation of a cytosolic cell death-inducing signalling complex.
The importance of cell death for intestinal homeostasis and inflammatory bowel disease (IBD) has been extensively studied and demonstrated using gene-targeted knockout mice. Loss of proteins such as NEMO, RIPK1, caspase-8 and FADD in the intestinal epithelium causes spontaneous intestinal pathology due to aberrant activation of inflammatory cell death [16,[19][20][21]. OTULIN deletion in the IECs does not cause spontaneous pathology, however sensitizes mice to experimental colitis induced by exposure to DSS, and increases the susceptibility to Salmonella infection, confirming the importance of OTULIN for barrier integrity in conditions of inflammation and infection. Similar to the phenotype of OTULIN IEC-KO mice, mice with intestinal epithelial deficiency in A20, a crucial ubiquitin-binding anti-inflammatory and cytoprotective protein that has been associated with IBD [40,41], do not develop spontaneous intestinal pathology, but are also hypersensitive to TNF and DSS [18] and to infection with Salmonella [38]. In addition, a recent study showed that mice that lack the C-terminal catalytic center of HOIP, abolishing LUBACs E3 ligase activity, specifically in IECs are also normal but sensitized to TNF and DSS, indicating that linear ubiquitination in IECs protects against intestinal inflammation by suppressing TNF-induced IEC apoptosis under inflammatory conditions [42]. Together, our study identifies OTULIN as a major antiapoptotic protein in the intestinal epithelium that ensures maintenance of epithelial barrier integrity in inflammatory conditions, and suggest that OTULIN defects may increase susceptibility to IBD. However, so far, no genetic associations have been reported for OTULIN in IBD.
Finally, although OTULIN has been identified as a negative regulator of TNF-induced NF-κB activation [7,9], especially in myeloid cell types, we did not observe a major inhibitory effect of OTULIN on homeostatic nor TNF-induced NF-κB signaling in IECs, in vivo nor in vitro, similar to earlier observations in fibroblasts, keratinocytes and hepatocytes [10,11,[13][14][15]30]. Based on these findings we can conclude that the intestinal phenotype of OTULIN IEC-KO mice is caused by the hypersensitivity of the OTULIN-deficient epithelium to cell death affecting barrier integrity.
All alleles were maintained on a C57BL/6 J genetic background. Mice were housed in individually-ventilated cages at the VIB Center for Inflammation Research, in a specific pathogen-free animal facility. All experiments on mice were conducted according to institutional, national and European animal regulations. Animal protocols were approved by the ethics committee of Ghent University (permit number LA1400091).

Isolation of intestinal crypts and three-dimensional organoid culture
Intestinal organoids were derived from the small intestine, as previously described [43]. Briefly, a 10 cm piece of duodenum/ jejunum was dissected and washed in phosphate-buffered saline (PBS). The intestine was opened longitudinally, villi were scraped away and the tissue was chopped in 2-3 mm pieces. After thorough washing in PBS, pieces were incubated in 2 mM EDTA/ PBS for 30 min at 4°C on a rocking platform. The pieces were shaken vigorously in 2% fetal calf serum and after passage through a 70-μm cell strainer, crypt fractions were isolated and purified by successive centrifugation steps. One ml of matrigel (BD Biosciences) was added to a pellet of 100-1000 crypts, and drops of 50 μl crypt-containing matrigel were added to pre-warmed wells in a 24-well plate. After polymerization, 500 μl complete growth medium (advanced DMEM/F12, Gibco) containing EGF (50 ng/mL, Invitrogen), R-Spondin1 (from conditioned medium from 293T-HA-RspoI-Fc cells), Noggin (from conditioned medium), N2 (Gibco), B2 (Gibco), Hepes (10 mM, Invitrogen), Glutamax (Invitrogen), N-Acetylcycsteine (1 mM, Sigma) and 1% penicillin/ streptomycin (Gibco) [44] was added to each well every 2 days. For TNF stimulation (Escherichia coli-derived recombinant protein produced by VIB-Protein service facility), 20 ng/mL mTNF was diluted in organoid growth medium.

PI-Annexin cell death analysis
After induction of cell death, cells were stained with annexin V-APC (BioLegend, Cat# 640941, 1:50) and propidium iodide (Sigma, Cat# 4170,2 µg/mL) for 15 min at room temperature in annexin V binding buffer (BD). Flow cytometry analysis was performed using the FACS Calibur (BD) and LSR II HTS (BD). Data were analysed with FlowJo v.10 software.

Live cell imaging
Small intestinal organoids were isolated as described above and seeded in μ-slide 8 well (iBidi). CellEvent™ Caspase-3/7 Green Detection Reagent (Invitrogen) was added to the medium according to the manufacturer's instructions. Three organoids per condition (technical triplicates) were followed over time at time intervals of 30 min and z-stack images were made using the Zeiss spinning disk. Images were processed using ImageJ. For each image, the total volume of green signal was determined and normalized to the total volume of the organoid.

IEC isolation
Small intestines or colons were dissected and flushed with cold PBS to remove fecal contents. The intestine was turned inside out, washed in cold PBS and incubated in 2 mM EDTA/HBSS at 37°C for 20 min. Next, the tubes were shaken vigorously and the supernatant containing the IECs was further processed for Western blot or qPCR.

Immunoprecipitation
Recombinant GST-UBAN was produced in BL21(DE3) cells. In brief, BL21(DE3) cells were transformed with the plasmid encoding GST-UBAN and protein expression was induced with 0.5 M IPTG. After 4 h, cells were collected and lysed in lysis buffer (20 mM Tris-HCl pH 7.5, 10 mM EDTA, 5 mM EGTA, 150 mM NaCl, 1 mM DTT supplemented with phosphatase and protease inhibitor cocktail tablets (Roche Diagnostics)), sonicated and cleared by centrifugation. After centrifugation, Triton-X100 (0.5% final concentration) was added to the supernatant, which was then transferred onto prewashed glutathione beads and left rotating for 2 h at 4°C. After incubation, the beads were centrifuged, washed twice with washing buffer (20 mM Tris-HCl pH 7.5, 10 mM EDTA, 150 mM NaCl, 0.5% Triton-X100) and resuspended in resuspension buffer (20 mM Tris-HCl pH 7.5, 0.1% β-mercaptoethanol, 0.05% sodiumazide), ready to be used. Cell lysates from IECs were prepared as described before, protein concentration was determined and 2 mg protein lysate was incubated overnight with GST-UBAN-containing glutathione beads. The beads were washed three times in RIPA lysis buffer (150 mM NaCl, 1% NP-40, 0.5% Sodium Deoxycholate, 0.1% SDS, 10 mM Tris-HCl pH 8 supplemented with phosphatase (Sigma) and protease (Roche Diagnostics) inhibitor cocktail tablets), and resuspended in 60 μL 1x laemmli buffer for direct analysis. For immunoprecipitation of complex I, HCT116 cells were stimulated with 1.5 μg/ml Flag-hTNF (VIB Protein Service Facility). Cells were then washed 2 times in icecold PBS and lysed in 1 ml NP-40 lysis buffer (10% glycerol, 1% NP-40, 150 mM NaCl and 10 mM Tris-HCl pH 8, supplemented with phosphatase and protease inhibitor cocktail tablets (Roche Diagnostics)). The cell lysates were cleared by centrifugation for 15 min at 4°C and the supernatants were then incubated overnight with FLAG M2 affinity gel (Sigma-Aldrich). Receptor complex proteins were eluted from the beads using 200 ng/mL 3× FLAG peptide (Sigma) as described in the manufacturer's instructions. Protein complexes were additionally deubiquitylated by incubation of beads with 1.2 μg USP21 (VIB Protein Service Facility) in DUB buffer (50 mM Tris-HCl pH8, 50 mM NaCl, 5 mM DTT and 1 mM MnCl2) for 30 min at 37°C before FLAG peptide elution. For immunoprecipitation of complex II, HCT116 cells were pretreated with ZVAD (50 µM, Enzo Life Sciences) for 30 min, followed by hTNF (20 ng/mL) stimulation for the indicated timepoints. The cells were lysed in NP40 lysis buffer and lysates were cleared by centrifugation and the supernatants were incubated with RIPK1 antibody (Cell signaling, Cat# 3493) and Protein A Sepharose beads (Cytiva, GE17-5280-01).
In vivo TNF toxicity Mice were injected intraperitoneally with a sublethal dose of mouse TNF (5 μg mouse TNF/20 g body weight). E. coli-derived recombinant mTNF had a specific activity of 9.46 ×10 7 IU/mg, and was produced and purified to homogeneity in our laboratory with endotoxin levels not exceeding 1 ng/mg protein. Body temperature and survival were monitored every hour. Mice were euthanized on the indicated timepoints.
DSS-induced colitis 9-15-week old OTULIN IEC-KO and littermate WT controls were used in DSS-induced colitis experiments. Acute colitis was induced by addition of 2 % DSS (36-50 kD; MP Biomedicals) to the drinking water for 6 days followed by regular distilled water. Body weight, occult or gross blood loss per rectum, and stool consistency were determined daily during the colitis experiment. Fecal blood was determined using Hemoccult SENSA (Beckman Coulter) analysis. The baseline clinical score was determined on day 0. In brief, no weight loss was scored as 0, weight loss of 1-5% from baseline as 1; 5-10% as 2; 10-20% as 3; and >20% as 4. For bleeding, a score of 0 was assigned for no blood, 2 for positive hemoccult, and 4 for gross bleeding. For stool consistency, a score of 0 was assigned for well-formed pellets, pasty and semiformed stools were scored as 2, and liquid stools as 4. The average of these scores resulted in a total clinical score ranging from 0 (healthy) to 4 (maximal colitis). To eliminate any diagnostic bias, mice were scored blindly.

Salmonella-induced colitis
Littermate-, sex-and age-matched mice were infected using the Salmonella enterica serovar Typhimurium (strain SL1344 or ΔSPI-1ΔSPI-2) model of colitis [36]. SPF mice were given a single dose of 20 mg streptomycin via oral gavage (100 μl of 200 mg ml −1 streptomycin dissolved in water) one day before infection. Mice were infected with 1 × 10 7 CFU per mouse resuspended in 1× PBS via oral gavage. Mouse body weight was measured daily and the percentage body weight was calculated as (daily weight)/(day 0 starting weight). At the indicated day after infection, intestinal tissue (ileum, cecum and colon) and the spleen were collected. For bacterial burden measurements and histology, luminal contents of the ileum (the last 5-6 cm of the distal end of the small intestine) and the colon were removed. Luminal content was retained in all cecal samples. Any attaching lymphatic tissue was removed from intestinal tissue before homogenization in 1 ml of 1× PBS. CFU per gram of tissue were calculated by plating serial dilutions of tissue homogenates on MacConkey agar containing streptomycin and MacConkey agar containing streptomycin and kanamycin. Tissue samples were excluded if the total burden fell below the limit of detection. Single strain infection tissue homogenates were plated solely on MacConkey agar containing streptomycin.

Histology
Small intestine and colon were dissected, fixed in 10 % formalin and embedded in paraffin. Sections were stained with Hematoxylin/Eosin (HE) or with specific stains or antibodies. For immunohistochemistry, following dewaxing, slides were incubated in antigen retrieval solution (Dako), boiled for 20 min in a PickCell cooking unit and cooled down for 3 h. Endogenous peroxidase activity was blocked by incubating the slides in 3% wv H 2 O 2 (Sigma). The blocking buffer contains 0.2% goat serum, 0.5% Fish skin gelatin and 2 % BSA in PBS. Subsequently, slides were incubated with anti-cleaved caspase 3 (1:300, Cell Signaling, Cat# 9661), anti-Ki67 (1:1000, Cell Signaling, Cat# 12202), or F4/80 (1:50, BioRad, Cat# MCA497G) primary antibodies overnight at 4°C. Next, the slides were incubated with biotin coupled goat anti-rabbit (1:500, Dako, Cat# E0432), and incubated in ABC solution (Vectastain ELITE ABC Kit Standard, Vector Laboratories). Peroxidase activity was detected by adding diaminobutyric acid (DAB) substrate (ImmPACT DAB Peroxidase (HRP) Substrate, Vector Laboratories), and slides were counterstained with hematoxylin (Sigma), dehydrated and mounted with Entellan (Merck). For the F4/80 staining, a similar protocol was followed, without antigen retrieval and using 5% goat serum in 1% BSA as blocking buffer, and biotin coupled goat anti-rat (1:500, Vector Laboratories, Cat# BA-9401) as secondary antibody. TUNEL kit (Millipore) was used to visualize death cells, according to manufacturer's instructions. For combined Alcian Blue and Periodic Acid-Schiff stainings (AB/Pas), dewaxed sections were incubated in AB for 20 min. Sections were subsequently washed with water before incubation in 1% periodic acid for 10 min followed by incubation in Schiff's reagent for 10 min. Sections were counterstained with Mayer's haematoxylin for 30 s, washed and dehydrated before mounting with Entellan (Merck). The pictures were taken with the Zeiss slide scanner. To eliminate any diagnostic bias, quantification of cleaved caspase 3 and TUNEL stainings was done blindly.
Histomorphologyical score of TNF emARE sections HE sections of TNF emARE mice were scored blindly according to following scoring scheme: for goblet cells a score of 0 was given if the amount of goblet cells is normal, 1 for minimal goblet cell loss (1-30 %), 2 for obvious goblet cell loss (30-60 %), 3 for severe goblet cell loss (60-90 %) and 4 for maximal goblet cell los (>90%). For immune cell infiltration a score of 0 represents no immune cell infiltration, 1 immune cell infiltration in the lamina propria, 2 infiltration in the mucosa, 3 infiltration in the submucosa and 4 transmural infiltration. Finally, villus architecture was scored as 0 for normal structures, 1 for minimal villus blunting, 2 for blunted villi with only 50% of crypt remaining, 3 for blunted villi with only 25% of crypt remaining and 4 for maximal blunting and loss of callus-crypt architecture. The three scores were summed resulting a score ranging from 0 (normal) to 12 (maximal pathology).

Quantification of cytokines
Serum was isolated from adult mice and IL-6 and Ccl2 was measured by Bio-Plex (Biorad), according to manufacturer's instructions. TNF levels were determined by ELISA (Mouse Uncoated ELISA Kit with Plates, Invitrogen) according to manufacturers' instructions.

Flow cytometry analysis
Lamina propria isolation from the small intestine of mice was performed by two treatments with HBSS + 2 mM EDTA, followed by treatment with collagenase VIII (0,6 mg/ml). The isolated cells ]. The cells were later fixed and processed for analysis using a five-laser BD LSRFortessa. The flow cytometry data were analyzed using FlowJo software 10.8.1 and presented as percentage to CD45+ population.

Quantification and statistical analysis
Results are expressed as the mean ± SEM (unless stated otherwise), with n the number of mice. Outliers were identified using graphpad (ROUT, Q = 1%) and excluded from analysis. Normality tests (Kolmogorov-Smirnov) were performed to check for normal distribution of the data. Parametric tests were performed on normally distributed data, and non-parametric tests on data that were not normally distributed. Statistical significance between WT and OTULIN IEC-KO was assessed using an unpaired two-sided Student t-Test, or non-parametric Mann-Whitney test. Statistical significance between WT and OTULIN IEC-KO at different time points or between untreated and Salmonella infected was assessed using a two-way ANOVA test, followed by Sidák's multiple comparison test. A Kruskal-Wallis test was performed for the analysis between WT, OTULIN IEC-KO , TNF emARE and OTULIN IEC-KO TNF emARE mice followed by a Dunn's multiple comparison test. Survival of mice after injection with TNF was analysed using a Mantel-Cox test. All above analysis were performed in the Prism software (V9). Statistical details can be found in the figure legends. Body temperature, body weight, clinical disease score and organoid live cell imaging data followed over time were analysed as repeated measurements using method of residual maximum likelihood (REML), as implemented in Genstat version 20. Briefly, a linear mixed model (random terms underlined) of the form y = genotype + time + genotype × time + mouse × time was fitted to the longitudinal data. The term mouse × time represents the residual error term with dependent errors because the repeated measurements are taken in the same individual, causing correlations among observations. The autoregressive correlation model (AR) was finally selected as best fitted model based on the Akaike's information criterion coefficient. The AR covariance model assumes that correlation between observations decays as the measurements are collected further apart in time. Additional options selected to get a best fitting model included 1) times of measurement were set as equally spaced, and 2) allowance of unequal variances across time. The significance of the fixed main and interaction terms in the model were assessed using an approximate F-test as implemented in Genstat version 20 [45].

DATA AVAILABILITY
All datasets generated and analysed during this study are included in this published article and its Supplementary Information files. Additional data are available from the corresponding author on reasonable request.